Project Summary Blood diseases such as anemias and hemophilia as well as cancers such as lymphoma, myeloma, and leukemia affect millions of Americans. Current treatment methods for these carry high levels of risk and are not entirely successful. Study of hematopoietic development is crucial to understanding how these diseases occur and in developing new more effective treatment methods. The nature of zebrafish development makes them an ideal model for studying hematopoietic cell development. In contrast with mammalian models, the external rapid development of zebrafish along with fluorescent reporter lines make visualization and tracking of developing blood and immune cells easy in the transparent embryos. In addition, unlike invertebrate models, the types of hematopoietic cells, their markers, and pathways are highly conserved from zebrafish to mammals. We have identified a group of cells by expression of hematopoietic markers scl and etv2. Etv2 is an ETS transcription factor that acts as a master regulator of hematopoiesis and vascular development that has function conserved among vertebrates. Scl is a bHLH transcription factor downstream of Etv2 that is one of the earliest required genes required for hematopoiesis. Because of the bilateral location of these cells just above the yolk extension adjacent to the pronephros we have named these cells pronephros-associated cells or PACs. Although at later stages, HSCs will colonize the pronephros, a functional equivalent of the mammalian bone marrow HSC niche, this domain does not correspond to any known hematopoietic site at this time point. From our preliminary data, we suggest that PACs can differentiate into different types of blood and immune lineages including macrophages and can also translocate to the vasculature. However, the origin and signaling pathways that specify these cells has not been identified. In addition their functional capabilities and contribution to hematopoietic lineages is unknown. Due to the highly conserved pathways and analogous hematopoietic sites between zebrafish and mammals, it is likely that these cells exist in mammalian embryos as well. We will utilize zebrafish to test the hypothesis that PACs are a population of uncharacterized hematopoietic progenitors that give rise to multiple blood cell types. We will determine the lineage contribution of PACs and expect that they will contribute to macrophage and other blood cell lineages. We will also characterize the expression profile of these cells compared to known hematopoietic stem and progenitor cells. The data derived from this study will help us better understand hematopoietic development and will aid in developing new treatments for life threatening blood diseases.